Isolation of RP-HPLC pure clonidine-displacing substance from NG108-15 cells

A crude extract of clonidine-displacing substance (CDS) has previously been extracted from the NG108-15 cell line. This study aimed to purify CDS extracted from this cell line further by the technique of reverse phase-HPLC (RP-HPLC), and subsequently determine whether this refined CDS bears any similarity to CDS's extracted from other tissues. Crude CDS was extracted from NG108-cells and fractionated by RP-HPLC eluting with a linear gradient of methanol (5-65%; 1 ml min(-1) flow rate) over 50 min., and collected at 1 min. intervals. The pharmacological activities of the CDS fractions were determined by their abilities to displace bound [H-3]clonidine to alpha(2)-adrenoceptors in mt brain membranes. RP-HPLC analysis of CDS revealed a pharmacologically active fraction distinct from agmatine, eluting at 24 min, corresponding to an absorbance peak observed at this time. Collectively, these results confirmed that CDS was present in the NG108-15 cell Line. However, the RP-HPLC analysis showed the pharmacological activity to elute at a more hydrophobic gradient than previously observed with CDS's extracted from bovine tissues. These results support the notion of the existence of several CDS's. (C) 2000 Elsevier Science B.V. All rights reserved.